转录因子Foxq1通过WNT/β-catenin信号通路影响绒山羊毛囊干细胞增殖的研究

doi:10.11843/j.issn.0366-6964.2023.06.041

摘要/Abstract

摘要： 旨在探究陕北白绒山羊毛囊诱导期(E 66)和分化期(E 120)皮肤组织差异表达基因Foxq1在绒山羊毛囊干细胞(HFSCs)中的功能。本研究选择年龄、体重基本一致的6只健康妊娠陕北白绒山羊母羊,在胚胎期E 66(n=3)和E 120(n=3)分别采集胎儿背部皮肤组织。将pAd- Foxq1和pAd-Blank过表达腺病毒分别侵染HFSCs,利用EdU、MTT及流式细胞等方法检测Foxq1对HFSCs增殖的影响;利用qPCR和免疫荧光试验检测Wnt信号通路激活的标记因子CTNNB1及Wnt信号通路下游基因的表达情况。qPCR结果表明,Foxq1在绒山羊E 66和E 120皮肤组织差异表达(P<0.001),与测序数据一致;荧光显微镜下观察到彗星尾状荧光出现,表明Foxq1腺病毒包装成功,且qPCR结果显示Foxq1的过表达效率高达40 000多倍(P<0.000 1)。EdU和MTT结果表明,过表达Foxq1后,毛囊干细胞的活力显著增强(P<0.05),EdU阳性细胞数量显著升高(P<0.01)。流式细胞周期结果显示,过表达Foxq1后S期细胞比率显著升高。qPCR结果表明,过表达Foxq1后CTNNB1、TCF3及CYCLIND1基因的mRNA表达量显著升高(P<0.05),免疫荧光进一步证明过表达Foxq1后CTNNB1、TCF3及CYCLIND1蛋白水平的表达量显著升高。本研究发现,Foxq1能够激活Wnt/β-catenin信号通路从而促进HFSCs的增殖,揭示了Foxq1通过WNT/β-catenin信号通路影响绒山羊毛囊干细胞增殖的分子机理。

关键词: Foxq1, 毛囊干细胞, Wnt信号通路, 细胞增殖, 绒山羊

Abstract: This study aimed to explore the function of Foxq1, a differentially expressed gene in the skin tissues at hair follicle induction stage (E 66) and cytodifferentiation stage (E 120), in hair follicle stem cells (HFSCs) of Shanbei white cashmere goat. In this study, 6 healthy pregnant Shanbei white cashmere goats with the same age and body weight were selected. And the back skin tissues were collected at E 66 (n=3) and E 120 (n=3) from the fetuses, respectively. The pAd-Foxq1 and pAd-Blank overexpression adenovirus vectors were transfected into HFSCs. EdU, MTT and flow cytometry were used to detect the effects of Foxq1 on the proliferation of HFSCs. qPCR and immunofluorescence assay were used to detect the expression of CTNNB1, a marker of activating Wnt signaling pathway, and the downstream genes of Wnt signaling pathway. The results of qPCR showed that Foxq1 was differentially expressed in the skin tissues of cashmere goats at E 66 and E 120 stages (P<0.001), which was consistent with the sequencing data. Comet tail fluorescence was observed under fluorescence microscope, indicating that Foxq1 adenovirus was successfully packed, and qPCR results showed that the overexpression efficiency of Foxq1 was more than 40 000 times (P<0.000 1). EdU and MTT results showed that the cell viability of hair follicle stem cells was significantly increased with the overexpression of Foxq1 (P<0.05), and the number of EdU positive cells was significantly increased (P<0.01). Flow cytometry results showed that the percentage of S-phase cells was significantly increased with the overexpression of Foxq1. qPCR results showed that the mRNA expressions of CTNNB1, TCF3 and CYCLIND1 genes were significantly increased with the overexpression of Foxq1 (P<0.05). Immunofluorescence result further demonstrated that the protein expression levels of CTNNB1, TCF3 and CYCLIND1 were significantly increased with the overexpression of Foxq1. This study revealed that Foxq1 could activate the Wnt/β-catenin signaling pathway to promote the proliferation of HFSCs, which illustrated the function and molecular mechanism of Foxq1 in the proliferation of HFSCs.

Key words: Foxq1, HFSCs, Wnt signaling pathway, cell proliferation, cashmere goat

中图分类号:

S827.2

许甜甜, 张彤彤, 王蒙, 王昕. 转录因子Foxq1通过WNT/β-catenin信号通路影响绒山羊毛囊干细胞增殖的研究[J]. 畜牧兽医学报, 2023, 54(6): 2653-2661.

XU Tiantian, ZHANG Tongtong, WANG Meng, WANG Xin. Transcription Factor Foxq1 Affects the Proliferation of Hair Follicle Stem Cells in Cashmere Goats via WNT/β-catenin Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2653-2661.

参考文献 40

[1]	JI S F,ZHU Z Y,SUN X Y,et al.Functional hair follicle regeneration:an updated review[J].Signal Transduct Target Ther,2021, 6(1):66.
[2]	ABREU C M,MARQUES A P.Recreation of a hair follicle regenerative microenvironment:Successes and pitfalls[J].Bioeng Transl Med,2021,7(1):e10235.
[3]	CHEN D M,JARRELL A,GUO C T,et al.Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation[J].Development,2012,139(8):1522-1533.
[4]	WANG J,QU J W,LI Y J,et al.miR-149-5p regulates goat hair follicle stem cell proliferation and apoptosis by targeting the CMTM3/AR axis during superior-quality brush hair formation[J].Front Genet,2020,11:529757.
[5]	SIMICEVIC J,DEPLANCKE B.Transcription factor proteomics—Tools,applications,and challenges[J].Proteomics, 2017,17(3-4): 1600317.
[6]	NEWMAN J A,AITKENHEAD H,GAVARD A E,et al.The crystal structure of human forkhead box N1 in complex with DNA reveals the structural basis for forkhead box family specificity[J].J Biol Chem,2020,295(10):2948-2958.
[7]	BRANCACCIO A,MINICHIELLO A,GRACHTCHOUK M,et al.Requirement of the forkhead gene Foxe1,a target of sonic hedgehog signaling,in hair follicle morphogenesis[J].Hum Mol Genet,2004,13(21):2595-2606.
[8]	SHIROKOVA V,BIGGS L C,JUSSILA M,et al.Foxi3 deficiency compromises hair follicle stem cell specification and activation[J].Stem Cells,2016,34(7):1896-1908.
[9]	LAY K,KUME T,FUCHS E.FOXC1 maintains the hair follicle stem cell niche and governs stem cell quiescence to preserve long-term tissue-regenerating potential[J].Proc Natl Acad Sci U S A,2016,113(11):E1506-E1515.
[10]	ZHAO J Z,LI H J,ZHOU R J,et al.Foxp1 regulates the proliferation of hair follicle stem cells in response to oxidative stress during hair cycling[J].PLoS One,2015,10(7):e0131674.
[11]	WU B J,HERBERT PRATT C,POTTER C S,et al.R164C mutation in FOXQ1 H3 domain affects formation of the hair medulla[J].Exp Dermatol,2013,22(3):234-236.
[12]	GENTILE P,GARCOVICH S.Advances in regenerative stem cell therapy in androgenic Alopecia and Hair Loss:Wnt pathway,growth-factor,and mesenchymal stem cell signaling impact analysis on cell growth and hair follicle development[J]. Cells,2019,8(5):466.
[13]	李凡,陈晓阳,杨国雨,等.Wnt/β-连环蛋白信号通路在哺乳动物毛囊发育中的调控作用[J].畜牧兽医学报,2019, 50(11): 2186-2194.LI F,CHEN X Y,YANG G Y,et al.The regulatory role of Wnt/β-catenin signaling pathway in the development of mammalian hair follicles[J].Acta Veterinaria et Zootechnica Sinica,2019,50(11):2186-2194.(in Chinese)
[14]	FENG Y,GUN S B.Melatonin supplement induced the hair follicle development in offspring rex rabbits[J].J Anim Physiol Anim Nutr (Berl),2021,105(1):167-174.
[15]	MOPARTHI L,PIZZOLATO G,KOCH S.Wnt activator FOXB2 drives the neuroendocrine differentiation of prostate cancer[J].Proc Natl Acad Sci U S A,2019,116(44):22189-22195.
[16]	KOCH S.Regulation of Wnt signaling by FOX transcription factors in cancer[J].Cancers (Basel),2021,13(14):3446.
[17]	BAGATI A,BIANCHI-SMIRAGLIA A,MOPARTHY S,et al.Melanoma suppressor functions of the carcinoma oncogene FOXQ1[J].Cell Rep,2017,20(12):2820-2832.
[18]	GE W,WANG S H,SUN B,et al.Melatonin promotes cashmere goat (Capra hircus) secondary hair follicle growth:a view from integrated analysis of long non-coding and coding RNAs[J].Cell Cycle,2018,17(10):1255-1267.
[19]	刘丽华,钟震宇,郑玉杰,等.lncRNA EPB41L4A-AS通过调控ErbB3抑制奶牛乳腺上皮细胞增殖的研究[J].畜牧兽医学报, 2022, 53(7):2152-2159.LIU L H,ZHONG Z Y,ZHENG Y J,et al.lncRNA EPB41L4A-AS inhibits the proliferation of bovine mammary epithelial cells by regulating the expression of ErbB3 gene[J].Acta Veterinaria et Zootechnica Sinica,2022,53(7):2152-2159.(in Chinese)
[20]	王妞.FGF5和FGF21影响绒山羊毛乳头细胞增殖及其调控机制研究[D].杨凌:西北农林科技大学,2022.WANG N.Study on the proliferation and regulation mechanism of FGF5 and FGF21 in dermal papilla cells in cashmere goats[D]. Yangling:Northwest A&F University,2022.(in Chinese)
[21]	HU X M,LI Z X,ZHANG D Y,et al.A systematic summary of survival and death signalling during the life of hair follicle stem cells[J].Stem Cell Res Ther,2021,12(1):453.
[22]	HENLEY M J,KOEHLER A N.Advances in targeting 'undruggable' transcription factors with small molecules[J].Nat Rev Drug Discov,2021,20(9):669-688.
[23]	WEIDEMVLLER P,KHOLMATOV M,PETSALAKI E,et al.Transcription factors:bridge between cell signaling and gene regulation[J].Proteomics,2021,21(23-24):e2000034.
[24]	CHRISTENSEN J,BENTZ S,SENGSTAG T,et al.FOXQ1,a novel target of the wnt pathway and a new marker for activation of Wnt signaling in solid tumors[J].PLoS One,2013,8(3):e60051.
[25]	EARLEY A M,DIXON C T,SHIAU C E.Genetic analysis of zebrafish homologs of human FOXQ1,foxq1a and foxq1b,in innate immune cell development and bacterial host response[J].PLoS One,2018,13(3):e0194207.
[26]	HONG H K,NOVEROSKE J K,HEADON D J,et al.The winged helix/forkhead transcription factor Foxq1 regulates differentiation of hair in satin mice[J].Genesis,2001,29(4):163-171.
[27]	CHEN C L,HUANG W Y,WANG E H C,et al.Functional complexity of hair follicle stem cell niche and therapeutic targeting of niche dysfunction for hair regeneration[J].J Biomed Sci,2020,27(1):43.
[28]	PLIKUS M V.New activators and inhibitors in the hair cycle clock:targeting stem cells' state of competence[J].J Invest Dermatol,2012,132(5):1321-1324.
[29]	ALBRECHT L V,TEJEDA-MUÑOZ N,DE ROBERTIS E M.Cell biology of canonical Wnt signaling[J].Annu Rev Cell Dev Biol,2021,37:369-389.
[30]	XING Y Z,MA X G,GUO H Y,et al.Wnt5a suppresses β-catenin signaling during hair follicle regeneration[J].Int J Med Sci, 2016,13(8):603-610.
[31]	HOWARD J M,NUGUID J M,NGOLE D,et al.Tcf3 expression marks both stem and progenitor cells in multiple epithelia[J]. Development,2014,141(16):3143-3152.
[32]	ZHANG L,REN C F,YANG Z,et al.Forkhead Box S1 mediates epithelial-mesenchymal transition through the Wnt/β-catenin signaling pathway to regulate colorectal cancer progression[J].J Transl Med,2022,20(1):327.
[33]	ZHANG J,ZHANG C X,SANG L,et al.FOXF2 inhibits proliferation,migration,and invasion of Hela cells by regulating Wnt signaling pathway[J].Biosci Rep,2018,38(5):BSR20180747.
[34]	PIZZOLATO G,MOPARTHI L,SÖDERHOLM S,et al.The oncogenic transcription factor FOXQ1 is a differential regulator of Wnt target genes[J].J Cell Sci,2022,135(19):jcs260082.
[35]	GAO Y H,HUANG E Y,ZHANG H M,et al.Crosstalk between Wnt/β-catenin and estrogen receptor signaling synergistically promotes osteogenic differentiation of mesenchymal progenitor cells[J].PLoS One,2013,8(12):e82436.
[36]	XIANG L S,ZHENG J M,ZHANG M D,et al.FOXQ1 promotes the osteogenic differentiation of bone mesenchymal stem cells via Wnt/β-catenin signaling by binding with ANXA2[J].Stem Cell Res Ther,2020,11(1):403.
[37]	PENG X D,LUO Z,KANG Q J,et al.FOXQ1 mediates the crosstalk between TGF-β and Wnt signaling pathways in the progression of colorectal cancer[J].Cancer Biol Ther,2015,16(7):1099-1109.
[38]	LIU Z H,QIN Y,DONG S X,et al.Overexpression of miR-106a enhances oxaliplatin sensitivity of colorectal cancer through regulation of FOXQ1[J].Oncol Lett,2020,19(1):663-670.
[39]	LIN Y,LIN F K,ANUCHAPREEDA S,et al.Effect of miR-133b on progression and cisplatin resistance of triple-negative breast cancer through FGFR1-Wnt-β-catenin axis[J].Am J Transl Res,2021,13(6):5969-5984.
[40]	付强,岳巧娴,锡建中,等.绵羊miR-127/FOXO4反馈环路及其对卵泡颗粒细胞凋亡相关基因表达的影响[J].畜牧兽医学报,2022,53(1):66-75.FU Q,YUE Q X,XI J Z,et al.Effect of oar-miR-127/FOXO4 feedback loop on genes associated with apoptosis of sheep granulosa cells[J].Acta Veterinaria et Zootechnica Sinica,2022,53(1):66-75.(in Chinese)